Field of the Invention
The invention is directed toward a molecular diagnostic device that can be used to characterize genomic material that may be present in a sample.
Related Background Art
In the biotechnological field, there is a need for rapid identification of organisms, such as bacteria and viruses, in a variety of samples (e.g., environmental and medical). For example, rapid characterization of genomic material isolated from a bacterium (i.e., identification of the species and/or strain of the bacterium) may be necessary to provide quality assurance for, e.g., a local water supply, a hospital (including hospitalized patients therein), or a food processing plant; i.e., it may be necessary to monitor various samples, including but not limited to samples of air, dust, water, blood, tissues, plants, foodstuffs, etc., for the presence of contaminating organisms, and to identify the contaminating organisms prior to consumption, exposure, and/or use by the public, or during use by the public, or in tissue or blood samples obtained from a patient or another member of the public.
Standard microbiological methods for identifying an organism, e.g., culturing and Gram-staining or testing of other biochemical properties, are imprecise and often cannot differentiate among different organisms, let alone different strains of an organism. More precise methods for identifying an organism are based on the genomic DNA of the organism. Two such methods of identification are the polymerase chain reaction (PCR), for which technological developments (e.g., automated inline PCR platforms) have increased its level of throughput and automation, and the newer method of waveform profiling.
Because PCR exponentially amplifies DNA, it can be used to detect small amounts of genomic material. However, because PCR requires primers that are specifically complimentary to sequences of the genomic material that are known and bracket a DNA locus of interest, PCR is limited in that it can only be used for the characterization of known organisms. In other words, the investigator is required to know or guess the identity of the organism (i.e., the appropriate pair of primers to use) prior to any attempts at detecting the organism. Another limitation of PCR is the inability of the investigator, without further study, to map and/or obtain sequence information about the amplified DNA (and consequently the isolated genomic material) other than information about the sequences complimentary to the two primers used in the analysis. Additionally, an automated inline PCR platform generally does not provide a means to further analyze (e.g., map) genomic material after it has been subjected to PCR. Further analysis (e.g., providing more certain identification of a species and/or strain) of the genomic material may be important and useful in, e.g., distinguishing a pathogenic strain from a nonpathogenic strain, detecting and providing the sequence of a new strain, choosing an appropriate antibiotic regimen, etc.
To overcome some of the limitations of PCR, methods of waveform profiling were developed (see, e.g., U.S. patent application Ser. Nos. 11/190,942 and 11/356,807, and Japanese Patent Application Publication Nos. 2003-334082 and 2003-180351). Waveform profiling methods provide ways to analyze and profile genomic material, e.g., DNA isolated from organisms, such as bacteria, without requiring the investigator to know the identity of the organism prior to detection.
Waveform profiling generally utilizes melting temperature analysis accomplished with the use of detectible (e.g., radioactive, fluorescent, chemiluminescent, etc.) agents (e.g., nucleotides, intercalators, etc.) that are incorporated into higher-order DNA structures generated by waveform profiling. As the temperature of the sample is increased, the higher-order structures dissociate and, e.g., lose fluorescence intensity (e.g., intercalated fluorescent agents dissociate). Plotting the rate of change of fluorescence intensity obtained by the dissociation of these higher-order structures as a function of increasing temperature produces a waveform unique to the genomic DNA of the organism and the utilized waveform primer, i.e., the dissociation of higher-order DNA structures at different melting temperatures (Tm) is observed and recorded to produce a characteristic “waveform profile” for each species (or strain) of organism, e.g., bacteria. Accordingly, waveform profiling can be used to distinguish between genomic DNA isolated from a first organism and genomic DNA isolated from a second organism using melting temperature analysis. However, waveform profiling does not provide, without further investigation, the map or sequence of the analyzed genomic material.
Since waveform profiling is a relatively new method, advances described herein can be used to increase its throughput and/or automation.
As discussed above, new technologies that increase the level of PCR throughput and automation have been developed. An example of one such technology is the use of microfluidic systems, including controller/detector interfaces for such microfluidic systems, as described in, e.g., U.S. Pat. Nos. 6,033,546; 6,238,538; 6,267,858; 6,500,323; and 6,670,153. These microfluidic systems, collectively referred to herein as automated inline PCR platforms, are well known in the art and are generally described herein.
Most automated inline PCR platforms utilize a disposable microfluidic chip that works with controller/detector interfaces for automated sample accession, microfluidic PCR reagent assembly, PCR thermal cycling, and optical detection spectroscopy. A microfluidic chip generally comprises a first plate with at least one micro-etched fluidic (microfluidic) inline reaction channel that can be bonded to a second plate, within which can be metal traces and a fluid reservoir. When the two plates are bonded together, each microfluidic reaction channel of the first plate can connect with a fluid reservoir of the second plate so that locus-specific reagents can be delivered through the fluid reservoirs to the microfluidic inline reaction channels.
Inline PCR begins when a capillary, or “sipper,” aspirates a sample droplet (which may or may not be a DNA sample droplet, i.e., a sample droplet comprising genomic material isolated from an organism) from, e.g., a microtiter plate, into at least one microfluidic inline reaction channel. After aspirating a sample droplet into a microfluidic inline reaction channel, the sipper can be moved to a buffer trough so that buffer is drawn into the microfluidic chip. Consequently, cross-contamination among sample droplets is minimized or eliminated since each sample droplet is separated from adjacent sample droplets by buffer spacers. Each sample droplet then moves along a microfluidic inline reaction channel and into a PCR assembly area of the chip, wherein the sample droplet becomes a sample plug by being mixed with PCR-required reagents, e.g., a primer pair, DNA polymerase, and dNTPs, and detectable agents, e.g., intercalators, etc. Optionally, buffer spacers can also be mixed with PCR-required reagents to serve as negative controls. After being mixed with PCR-required and detectable agents, a sample plug (which may or may not be a DNA sample plug, i.e., a sample plug comprising genomic material) moves along the length of the microfluidic inline reaction channel into different areas of the chip, e.g., an amplification area wherein PCR can be effected on the sample plugs.
Generally, as each sample plug (e.g., a DNA sample plug) flows through a microfluidic inline reaction channel, it enters a temperature-controlled amplification area wherein each microfluidic inline reaction channel is repeatedly and rapidly heated and cooled in a localized manner such that the denaturing, annealing and elongation steps of PCR are effected on the sample plugs as they move through the channel(s); sample plugs that do not comprise genomic material are exposed to the same heating and cooling processes, etc. Amplification of DNA will occur only in DNA sample plugs, i.e., sample plugs comprising genomic material. A method of controlling the temperature in the amplification area is Joule heating (see, e.g., U.S. Pat. Nos. 5,965,410 and 6,670,153). Generally, voltage can be applied to the metal traces in or near the microfluidic inline reaction channel in a controlled and localized manner to effectuate the different temperatures required for each PCR cycle. Cooling of the reaction can be achieved through the use of, e.g., cooling fluid that travels through a coil to carry away thermal energy from the microfluidic inline reaction channel, rapid heat dissipation, e.g., by application of cold water to the bottom surface of the microfluidic chip, or simple radiant convection into the atmosphere or suitable heat transfer using a heat sink. Since the volume of fluid in the microfluidic channels is small and the metal traces are located very close to the microfluidic inline reaction channels, heating and cooling of the fluid in the channels (and hence, sample plugs) is accomplished very rapidly. Consequently, DNA sample plugs undergo PCR, and PCR cycles run such that, e.g., 30 cycles can typically be performed in, e.g., less than nine minutes. The number of PCR cycles each DNA sample plug sees as it travels through a microfluidic channel in the temperature-controlled area of the chip can be varied by changing, e.g., either or both 1) the timing of the voltage applied to the metal traces, and 2) the flow rate of the DNA sample plugs through the microfluidic channels.
A microfluidic chip can simultaneously perform as many polymerase chain reactions as it has microfluidic inline reaction channels. For example, a sample comprising genomic material can be aspirated into multiple different microfluidic inline reaction channels, to each of which is added a different locus-specific reagent (e.g., a different primer pair that brackets a different locus on the genomic material, e.g., DNA). This permits simultaneously detecting several different loci of genomic material isolated from the same organism. Alternatively, reagents comprising one specific primer pair can be aspirated into multiple different microfluidic inline reaction channels. This permits simultaneously detecting the same locus on genomic material isolated from different samples and/or different organisms. Additionally, multiple sample droplets can be aspirated into the same microfluidic reaction channel.
A detection area is usually downstream of the temperature-controlled amplification area, and is generally a transparent region that facilitates observation and detection of the amplified DNA products, e.g., PCR products. In the detection area, each microfluidic inline reaction channel is usually brought in close proximity and passed under a detector. A light source spreads light across the microfluidic inline reaction channels so that detectable agents or energy, e.g., fluorescence emitted from each channel, e.g., from each DNA sample plug, passing through the optical detection area can be measured simultaneously. After detection, each microfluidic inline reaction channel usually directs each sample plug to a waste well.
Three different methods are usually used to generate fluid motion within microfluidic inline reaction channels; the methods involve electrokinetics, pressure, or a hybrid of the two (see, e.g., U.S. Pat. Nos. 6,238,538; 6,670,153; 6,787,088; and U.S. Published Patent Application No. 2001/0052460) and nonmechanical valves (see, e.g., U.S. Pat. Nos. 6,681,788 and 6,779,559). In a pressure-based flow system, an internal or external source can be used to drive the flow of fluid in the inline reaction channels. For example, a vacuum can be applied to waste wells at the ends of each microfluidic inline reaction channel and can be used to activate the sipper and move the fluid along the microfluidic inline reaction channels toward the waste wells. Alternatively, since genomic material is charged, electrokinetics, i.e., the generation of a voltage gradient (e.g., by the application of voltage to the metal traces) can be used to drive charged fluid along the microfluidic inline reaction channels. A third method of driving the fluid along the inline reaction channels uses both electrokinetics and pressure. The result is a continuous flow of fluid within the microfluidic inline reaction channels, wherein sample plugs (e.g., DNA sample plugs) are continuously being mixed or moved to different areas (e.g., a PCR assembly area, a temperature-controlled area, a detection area, etc.) of the chip.
Electrokinetic and/or pressure-driven fluid movement, heating and cooling cycles, detection, and the data acquisition related to a microfluidic chip can be controlled by an instrument that interfaces with the chip (generally described in, e.g., U.S. Pat. Nos. 6,033,546 and 6,582,576). The interface of the instrument usually contains o-ring seals that seal the reagent wells on the chip, pogo pins that can interface with the metal traces on the chip and supply the voltage for temperature cycling, o-ring seals for the waste wells where a vacuum can be applied to move the fluid through the chip, a large o-ring that can be used to seal the bottom of the chip against circulating cool water and to speed the cooling during the temperature cycling, and a detection zone for, e.g., fluorescence detection. The risk of contamination with this system is minimal because a microfluidic chip is usually a closed system with physical barriers (e.g., buffer spacers) separating DNA sample plugs. Moreover, continuous flow prevents sample plugs from moving backwards.
The automated inline PCR platforms described above are limited in that the microfluidic chips should be disposed of after use and are not suitable for automated inline waveform profiling; also, analyzing samples using such a platform requires outsourcing. Additionally, the use of a sipper to aspirate sample droplets is an inefficient and wasteful method to obtain the small volume required to effect PCR cycles rapidly. The present invention resolves these limitations by providing a molecular diagnostic device that can be used to characterize genomic material isolated from an organism (e.g., bacteria, viruses) in a sample by automated methods of preparing the genomic material, and then either or both 1) amplifying the genomic material and detecting any amplified products and 2) mapping the genomic material. A molecular diagnostic device of at least one exemplary embodiment of the invention has the advantage that multiple samples, e.g., patient samples, can be processed through the same microfluidic chip without cross-contamination. Also, because in some exemplary embodiments the device is a portable system, a device of at least one exemplary embodiment of the invention can be utilized at different patient care centers throughout the Unites States or elsewhere in the world, and can also be used in a near-patient setting or a contaminated site away from a hospital or other patient care center. The molecular diagnostic device disclosed herein also has the advantage of facilitating the screening of samples within a short time after collection.